Citrus variegated chlorosis (CVC) is caused by Xylella fastidiosa, a phytopathogenic bacterium that can infect all Citrus sinensis cultivars. The endophytic bacterial communities of healthy, resistant, and CVC-affected citrus plants were studied by using cultivation as well as cultivation-independent techniques. The endophytic communities were assessed in surface-disinfected citrus branches by plating and denaturing gradient gel electrophoresis (DGGE). Dominant isolates were characterized by fatty-acid methyl ester analysis as Bacillus pumilus, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium spp. (including Methylobacterium extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, and M. zatmanii), Nocardia sp., Pantoea agglomerans, and Xanthomonas campestris. We observed a relationship between CVC symptoms and the frequency of isolation of species of Methylobacterium, the genus that we most frequently isolated from symptomatic plants. In contrast, we isolated C. flaccumfaciens significantly more frequently from asymptomatic plants than from those with symptoms of CVC while P. agglomerans was frequently isolated from tangerine (Citrus reticulata) and sweet-orange (C. sinensis) plants, irrespective of whether the plants were symptomatic or asymptomatic or showed symptoms of CVC. DGGE analysis of 16S rRNA gene fragments amplified from total plant DNA resulted in several bands that matched those from the bacterial isolates, indicating that DGGE profiles can be used to detect some endophytic bacteria of citrus plants. However, some bands had no match with any isolate, suggesting the occurrence of other, nonculturable or as yet uncultured, endophytic bacteria. A specific band with a high G؉C ratio was observed only in asymptomatic plants. The higher frequency of C. flaccumfaciens in asymptomatic plants suggests a role for this organism in the resistance of plants to CVC.

The diversity of endophytic bacterial populations of potato (Solanum tuberosum cv Desirée) was assessed using a combination of dilution plating of plant macerates followed by isolation and characterization of isolates, and direct PCR-DGGE on the basis of DNA extracted from plants. The culturable endophytic bacterial communities detected in potato stem bases as well as in roots were in most cases on the order 103 to 105 CFU g?1 of fresh plant tissue. Dilution plating revealed that a range of bacterial types dominated these populations. Dominant isolates fell into the a and g subgroups of the Proteobacteria, as well as in the Flavobacterium/Cytophaga group. Different representatives of the Firmicutes were also found. The most frequently isolated strains (>5% of the total) were characterized as different Pseudomonas spp. (including P. aureofaciens, P. corrugata, and P. putida), Agrobacterium radiobacter, Stenotrophomonas maltophilia, and Flavobacterium resinovorans, using fatty acid methyl ester (FAME) analysis and/or sequencing of their partial 16S ribosomal RNA genes. Other Proteobacteria or Firmicutes were also found, albeit infrequently, and mainly in potato stem tissue. The fate of three putative potato endophytes, Stenotrophomonas maltophilia, Bacillus sp., and Sphingomonas paucimobilis, was monitored following their release into potato plants via injection, via root dipping, or via the soil. Following stem injection, the S. maltophilia and Bacillus inoculants could be tracked over time periods of, respectively, 22 and 1 day(s) by dilution plating as well as via PCR-DGGE. However, only S. maltophilia was able to colonize, and persist in, plant tissue from soil or dipped roots. S. paucimobilis was never recovered from the plant irrespective of the mode of introduction. The diversity of the indigenous bacterial flora associated with potato was then monitored via PCR-DGGE. The patterns obtained revealed the existence of bacterial communities of limited complexity, with communities from potato stems typically differing from those from stem peel and roots. Evidence was obtained for the endophytic occurrence of a range of organisms falling into the a, b, and g subgroups of the Proteobacteria as well as in the Firmicutes. Several of the sequences found matched those from isolates, suggesting that the molecular evidence reported culturable organisms. However, a number of sequences did not have matching sequences from isolates, suggesting that non-culturable or as-yet-uncultured endophytic organisms were being detected.

Pea plants grown in the field were used to study the natural incidence of endophytic bacteria in the stem. Eleven pea cultivars at the flowering stage were screened for the presence of endophytic bacteria using a printing technique with surface disinfested stem cross-sections on 5% Trypticase Soy Agar (TSA). Five cultivars showed colonization. Cultivar Twiggy showed the highest and most consistent colonization and was further investigated. Stems of cv. Twiggy at the pod stage were analyzed for endophytic bacterial types and populations. Cross-sections of surface disinfested stems were printed on 5% TSA. Endophytic bacterial populations decreased from the lower to the upper part of the stem. One section from the third and the fourth internode was surface disinfested, homogenized, and spiral plated on the media 5% TSA, R2A, and SC (Davis et al. 1980). Over a series of 30 samples, 5% TSA gave significantly better recovery of bacterial endophytes compared with R2A and SC media. For most stems, populations ranged from 10(4) to 10(5) CFU/g except in one of the field blocks in which endophyte populations were uniformly higher. Comparison of colony counts by spiral plating and printing showed a positive correlation. The most frequently recovered bacterial types were Pantoea agglomerans and Pseudomonas fluorescens. Less frequently isolated were Pseudomonas viridiflava and Bacillus megaterium.

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Pea plants grown in the field were used to study the natural incidence of endophytic bacteria in the stem. Eleven pea cultivars at the flowering stage were screened for the presence of endophytic bacteria using a printing technique with surface disinfested stem cross-sections on 5% Trypticase Soy Agar (TSA). Five cultivars showed colonization. Cultivar Twiggy showed the highest and most consistent colonization and was further investigated. Stems of cv. Twiggy at the pod stage were analyzed for endophytic bacterial types and populations. Cross-sections of surface disinfested stems were printed on 5% TSA. Endophytic bacterial populations decreased from the lower to the upper part of the stem. One section from the third and the fourth internode was surface disinfested, homogenized, and spiral plated on the media 5% TSA, R2A, and SC (Davis et al. 1980). Over a series of 30 samples, 5% TSA gave significantly better recovery of bacterial endophytes compared with R2A and SC media. For most stems, populations ranged from 10(4) to 10(5) CFU/g except in one of the field blocks in which endophyte populations were uniformly higher. Comparison of colony counts by spiral plating and printing showed a positive correlation. The most frequently recovered bacterial types were Pantoea agglomerans and Pseudomonas fluorescens. Less frequently isolated were Pseudomonas viridiflava and Bacillus megaterium.

Field releases of the wild-type plant growth-promoting rhizobacterium Pseudomonas fluorescens 89B-27, its bioluminescent derivative GEM-8 (89B-27::Tn4431), and a spontaneous rifampin-resistant variant (R34) were established on cucumber in 1994 and 1995 to examine the efficiency of these marker systems for estimating the wild-type population. Seed and root samples were taken 0, 7, 14, 21 or 28, 35 or 42, and 70 days after planting in each year and processed for enumeration by spiral plating or immunofluorescent colony staining (IFC). In both years, the populations of 89B-27, R34, and GEM-8, as measured by IFC, were not significantly different (P > 0.05) from each other at each sampling time. However, the populations of R34 and GEM-8, as measured by spiral plating and differentiation based on their respective phenotypes, were significantly lower (P < 0.05) than the wild-type populations and their IFC-determined populations. These data indicate that traditional marker systems may underestimate populations and hence the survival and colonization of genetically marked bacteria.

Purothionins (PTHs) and hordothionins (HTHs) were purified by cation-exchange chromatography from petroleum-ether extracts of wheat and barley flour respectively. The HTHs could be separated into two fractions, HTH-1 and HTH-2. Radial diffusion assays and micro-plate broth dilution assays with a number of plant pathogenic bacteria showed that these proteins were toxic for Clavibacter michiganensis subsp, michiganensis, the causal agent of bacterial canker on tomato, C. m. subsp. sepedonicus, the causal agent of ring rot on potato, and Xanthomonas campestris pv. vesicatotqa, the causal agent of a spot disease on tomato and pepper. Only minor differences in toxicity between PTHs and HTHs, and between HTH-1 and HTH-2, were detected. Minor differences in toxicity of these thionins were also detected for different strains of these bacteria. The use of these plant proteins for engineering bacterial disease resistance into solanaceous crops will be discussed.

Immunomagnetic separation (IMS) procedures for the selective separation of Erwinia carotovora subsp. atroseptica from potato peel extract were optimized for the recovery of target and removal of non-target bacteria. A streptomycin-resistant strain of Erw. carotovora subsp. atroseptica was used in combination with a crystal violet pectate (CVP) medium supplemented with 100 micrograms ml-1 of streptomycin to determine the recovery level of the target bacterium. Recovery obtained with a polyclonal antiserum against Erw. carotovora subsp. atroseptica at a concentration of 6 micrograms IgG ml-1 was greater than that obtained with two monoclonal antibodies against lipopolysaccharides of Erw. carotovora subsp. atroseptica at a concentration of 10 micrograms IgG ml-1. A linear relationship was found between particle concentration ranging from 12 to 200 micrograms ml-1 and recovery level. When the Advanced Magnetics (AM) protein A and anti-rabbit IgG particles in the AM separation system and the Dynal anti-rabbit IgG particles in the Dynal separation system were examined, the highest recovery level per microgram of particles (66%) was obtained with the Advanced Magnetics protein A particles, followed by AM anti-rabbit particles (37%). Without IMS, detection of Erw. carotovora subsp. atroseptica in tuber peel extracts on a CVP-medium without streptomycin was impossible when the ratio of Erw. carotovora subsp. carotovora to Erw. carotovora subsp. atroseptica was greater than 100 or when large numbers of other saprophytic bacteria were present, because of overcrowding. IMS, using the AM anti-rabbit IgG particles, ensured that Erw. carotovora subsp. atroseptica could be enumerated in tuber peel extract consistently, to a detection level of 100 cells ml-1. Similarly, the IMS procedure lowered the detection level of Erw. carotovora subsp. atroseptica in a twofold diluted peel extract by PCR to ca 2.0 x 10(3) cells ml-1 or 50 cells per reaction tube. In contrast, positive results in PCR without IMS were obtained only when the peel extract was diluted 100 times and when the concentration of Erw. carotovora subsp. atroseptica was at least 10(5) cell ml-1.